ABSTRACT The increasing prevalence of drug-resistance among pathogenic bacteria to common antibiotics has become one of the most pressing global healthcare problems in modern society. There is an urgent need for novel antimicrobial agents against multidrug resistant (MDR) bacteria. Because the major cause of resistance is beta-lactamase (a bacteria-specific enzyme) to degrade antibiotics, the proposed work will turn the cause of resistance to the event of killing the bacteria. That is, this research will develop the precursors that are responsive to beta-lactamase for in-situ (i.e., on the surface of bacteria) formation of nanoscale assemblies that kill MDR bacteria. The goal of this work is to explore enzyme-instructed self-assembly?that is, the integration of enzymatic catalysis and molecular self-assembly?as a paradigm-shifting approach for the discovery and early development of novel therapies for treating infections caused by drug resistant bacteria. This proposal is both hypothesis and design driven. We hypothesize that enzyme-instructed self-assembly, as a unique way to localize the nanoscale assemblies of cationic peptides onto bacteria, will kill MDR bacteria. Our past results?beta-lactamase instructing self-assembly of peptides, self-assembled nanofibers of ultrashort cationic peptides inhibiting biofilms, enzyme-instructed self-assembly inhibiting bacterial growth, and multivalent antibiotics inhibiting MDR bacteria?strongly support the hypothesis. To validate the hypothesis, we will design cationic peptides that self-assemble upon the action of beta-lactamase, characterize the physiochemical properties of the peptides and their assemblies, and assess the antibacterial activities of the nanoscale assemblies against MDR bacteria. Specifically, we will evaluate 1) the effects of charge balance of the precursors, 2) the effects of side chain length of the cationic peptides, and 3) the effects of peptide sequence of the cationic peptides on the nanoscale assemblies of cationic peptides for killing MDR bacteria. By designing, synthesizing, and characterizing the ?-lactam containing precursors of cationic peptides and evaluate the antibacterial activities of the corresponding assemblies of the cationic peptides against MDR bacteria, we anticipate that this research will improve fundamental understanding of antimicrobial therapy, provide guiding principles to design antibacterial agents, and ultimately lead to an unprecedented approach of antibacterial drug development that integrates enzymatic reactions and molecular self-assembly.